Transmissible spongiform encephalopathies (TSEs or prion diseases) are a group of rare neurodegenerative diseases which include Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep, bovine spongiform encephalopathy (BSE), and chronic wasting disease (CWD) in mule deer and elk. TSE infectivity can cross species barriers. The fact that BSE has infected humans in Great Britain and concerns that CWD may act similarly in the US underscores the importance of understanding TSE pathogenesis and developing effective therapeutics. The infectious agent of TSE diseases is called a prion and is largely composed of an abnormally refolded, protease resistant form (PrPSc) of the normal, protease-sensitive prion protein, PrPC. Susceptibility to infection can be influenced by amino acid homology between PrPC and PrPSc while differences in structure between PrPSc molecules are believed to encode strain phenotypes. My laboratory addresses many different aspects of prion diseases at both the molecular and pathogenic level. In particular, my studies focus on: 1) identifying the earliest events which occur during prion infection, 2) defining the molecular pathways involved in prion-associated neurodegeneration, 3) determining the molecular basis of prion strains, 4) determining how PrPC sequence influences PrPSc formation and disease phenotype and, 5) development of effective prion therapeutics. Sporadic Creutzfeldt-Jakob disease (sCJD) is the most prevalent of the human prion diseases. The origin of sCJD is unknown, although the initiating event is thought to be the stochastic misfolding of endogenous PrPC into infectious PrPSc. By contrast, iatrogenic CJD (iCJD) is associated with exposure to an exogenous source of PrPSc. In both sCJD and iCJD, heterozygosity at residue 129 for methionine (M129) or valine (V129) in the prion protein may affect disease phenotype, onset, and progression. However, the relative contribution of each PrPC allotype to PrPSc in heterozygous cases of CJD is unknown. In 2016, we published mass spectrometry studies demonstrating that the relative abundance of PrPSc with M129 or V129 was highly variable in MV cases of sCJD but was dominated by PrPSc with V129 in cases of MV iCJD. Surprisingly, the PrPSc allotype ratio had no correlation with CJD type, age at clinical onset, or disease duration. Amino acid mismatches between PrPC and PrPSc influence the rate at which PrPSc forms and heterozygosity at key residues has the potential to significantly slow or even prevent disease transmission J Gen Virol 93: 2749-2756 (2012). In human PrPC, heterozygosity at codon 129 influences prion transmission and is a known resistance factor to sCJD ACTA Neuropathol 130: 159-170 (2015). Thus, it is possible that the PrPSc allotype ratio in heterozygous cases of CJD correlates with the efficiency of transmission of prion infectivity. In 2016, we inoculated transgenic mice overexpressing human PrPC with M129 with brain material from heterozygous cases of CJD with variable ratios of PrPSc M129 or V129. In tandem, we also initiated experiments to test the inoculated samples for their ability to promote the formation of PrPSc in vitro. These experiments will determine the influence of the PrPSc allotype ratio on prion disease tempo and transmission and will provide important insights into the mechanisms underlying CJD progression in humans. In 2016, we continued in vivo work to look at early events during prion infection following intracranial inoculation. These studies are designed to provide critical information about the events that occur during the first few hours following exposure to prions, including when formation of new PrPSc occurs and how PrPC is affected. Our most recent data have shown that the cellular location and biochemical properties of the host PrPC molecule change substantially during the first few hours post-infection. A manuscript describing these results is in preparation. Although there is an increasing body of work suggesting that mitochondrial dysfunction plays a role in several neurodegenerative diseases, the role of mitochondria in prion pathogenesis is poorly understood. In a paper we recently published that is described in our 2014 Annual Report J. Proteome Res. 13: 4620-4634 (2014), our proteomic analysis of two different mouse prion models suggested that mitochondrial pathways of apoptosis were involved the neurodegeneration associated with non-amyloid prion disease. In 2016, we have continued studies looking at how mitochondrial viability and bioenergetics change during the course of disease in multiple models of murine prion infection. Using in vivo and in vitro models of mitochondrial function, our data show that there is significant mitochondrial dysfunction at the clinical stages of prion disease. Furthermore, they suggest a role for PrPC in the mitochondria of healthy young mice. A manuscript on these studies has been submitted and another is in preparation. Our studies are the first to look at mitochondrial function throughout the course of prion disease and have the potential to identify new targets for therapeutic intervention. Finally, in 2016 we initiated studies to determine how post-translational modifications may influence PrPSc formation and prion pathogenesis in different mouse models of prion infection. Using our new 6550 iFunnel QTOF single quadrupole LC mass spectrometer, we are analyzing post-translational protein modifications such as phosphorylation, acetylation, oxidation etc. in prion-infected versus uninfected mice. In addition, we are studying how cell-specific differences in PrPC glycosylation may influence PrPSc formation and prion infection of cells. These studies will help to elucidate how post-translational modifications contribute not only to PrPSc formation but also to different prion disease phenotypes in vivo.